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CNS 7056X, an ultra-short acting benzodiazepine: in vitro ligand binding profile

Gavin J Kilpatrick¹, Margaret S McIntyre², Richard F Cox², Jeffrey A Stafford², Gregory J Pacofsky² and Gary S. Tilbrook¹.

¹CeNeS Limited, Compass House, Vision Park, Histon, Cambridge, Cambs, United Kingdom CB4 9ZR. ²GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA .

Introduction: A drawback with the clinical use of midazolam is the prolonged and variable duration of action. In an effort to improve on midazolam and building on the technology established with remifentanil, a range of benzodiazepine ligands have been designed that are rapidly metabolised by non-specific tissue esterases. The experiments outlined herein were conducted to evaluate the affinity of CNS 7056X and its carboxylic acid metabolite, CNS 7054X, to bind to the benzodiazepine site of the GABA_A receptor in brain tissue from rats, pigs and human. The affinity of these compounds for a range of other receptors, enzymes and ion channels is also described.

Methods: Human cerebral cortex membranes, Yucutan micropig cerebral cortex membranes and rat (Sprague-Dawley) whole brain membranes (minus cerebellum) were obtained from Analytical Biological Services Inc., Wilmington, Delaware USA. Membranes were prepared using method of Marangos and Martino (1). Stock membrane was diluted with assay buffer (50 mM TRIS hydrochloride, pH 7.4, 150 mM NaCl at 4^oC) to a final concentration of 15 mg per well. Plates were allowed to incubate for 90 minutes with [³H]-Flunitrazepam (2.5nM), 4^oC (final volume 200ul). Binding was terminated by filtering the contents of each plate through GF/B Unifilter 96 well microplates. Plates were washed with 8 times the well volume using ice cold TRIS HCl buffer, pH 7.4. Radioactivity was measured using a scintillation counter. Data were analysed using the RoboSage Microsoft Excel add-in. The selectivity profile of CNS 7056X and CNS 7054X were determined using a  Panlabs  screen (MDS Pharma services).

Results: CNS 7056X shows a high binding affinity for the benzodiazepine site of the GABA_A receptor. The Ki for competition with the reference ligand, [3H]flunitrazepam, binding to tissue homogenates from human, rat and pig brain (Table 1) was approximately 30nM (pKi  7.5). The acid metabolite of CNS 7056X, CNS 7054X, showed a markedly lower affinity, with Ki values of approximately 10,000nM (pKi  5.0). The separation between the affinity of CNS 7056X and its metabolite, CNS 7054X ranged from 410 fold in human brain tissue to 320 fold in rat brain tissue. The potential for side-effect liability was assessed by determining the affinity of CNS 7056X and CNS 7054X (10uM) for a range (40) of receptors and ion channels. These studies revealed no detectable affinity at any site other than the GABA_A receptor.

Conclusions: CNS 7056X is a high affinity, selective benzodiazepine ligand acting at the GABA_A receptor. A similar high affinity was observed using homogenates of human, rat or pig brain. The carboxylic acid metabolite, CNS 7054X, revealed a much lower affinity. The separation in affinity between CNS 7056X and CNS 7054X was greater than 300 fold, with the highest separation being seen in human tissue. Neither CNS 7056X nor CNS 7054X showed measurable affinity for any site tested other than the GABA_A receptor.

Table 1. Affinities of CNS 7056X and CNS 7054X to compete for [³H]flunitrazepam binding to brain homogenates

Results are the mean ± s.e.m. of 4-8 separate experiments

1.       Marangos PJ and Martino AM, Mol. Pharm. 20, 16. (1981).